Coaxial tubular sequestering device for micro spheres and cells

ABSTRACT

A device for the separation of small particles or cells from a fluid suspension of the same is described. The device includes a coaxial tubular design in which the inner tube is a micro porous tube that allows the passage of liquids and certain particulates up to a certain size cut-off, and the outer tube allows for the collection of passed fluids. Inlet and outlet ports allow the introduction and flushing of components of interest. Embodiments of the device can be used for the separation of blood components, the sequestering of micro spheres used in micro-sphere-based immuno assay, and sample filtration. Other applications are not precluded. Another field of application for this device is in the separation of plasma from red blood cells. The red blood cells will not pass through the membrane due to their size, but plasma will.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of non-provisional application Ser. No.11/308,207, filed Mar. 12, 2006 and now U.S. Pat. No. 7,625,762, whichis based on provisional Application No. 60/594,134, filed Mar. 14, 2005.

BACKGROUND OF THE INVENTION

The present invention relates to micro spheres. More specifically, theinvention relates to sequestering of micro spheres.

Micro spheres are used extensively to capture analytics of interest forvarious selective assays. For example, micro-sphere-based immuno-assayallows a means of conducting the determination of multiple analytessimultaneously in a single reaction vessel. Molecular reactions takeplace on the surface of tiny spheres called micro spheres or beads. Foreach reaction, target molecules are selectively attached to the surfaceof internally color-coded micro spheres. The assigned color codeidentifies the reaction throughout the test. The concentration of targetmolecules is measured using a second kind of molecule called a reporter,which attaches to the target molecule in a sandwich-type reaction.

Early attempts at developing a suitable sequestering cell sufferedbecause of a large pressure drop across the bead barrier. Either thesurface area of a porous membrane was too small or where a gap of ca 3micrometers (μm) was machined, the length of the bead barrier was tooshort and gave rise to a large back pressure in the sequestering cell.This problem is remedied by the present invention.

SUMMARY OF THE INVENTION

The present invention, in a first aspect, provides a tubularsequestering device for micro spheres, the tubular sequestering devicecomprising:

a porous tube having an internal volume of from about five to about ninemicroliters, and pores having a pore size with an appropriate dimensionto prevent passage of the micro spheres but allow passage of a liquid,for retaining the micro spheres within the tube while enabling a liquidcarrier in which the micro spheres are suspended to pass through thepores of the tube.

In a second aspect, the invention provides a sequestering device formicro spheres, the sequestering device comprising:

first and second passageways for a liquid carrier or for a suspension ofthe micro spheres in the liquid carrier; and

a planar porous disk, disposed between and fluidly connected to thefirst and second passageways, the porous disk including pores having apore size with an appropriate dimension to prevent passage of the microspheres but allow passage of a liquid, for retaining the micro sphereswithin the disk while enabling the liquid carrier in which the microspheres are suspended to pass through the pores of the porous disk.

In a third aspect, the invention provides a method for sequesteringmicro spheres, the method comprising:

using a porous membrane having a pore size with an appropriate dimensionfor retaining micro spheres suspended in a liquid carrier while passingthe liquid carrier through the porous membrane.

In a fourth aspect, the invention provides a method for sequesteringmicro spheres, the method comprising the steps of:

-   -   a. aspirating, with a pump, a suspension of the micro spheres in        a liquid carrier;    -   b. using a holding coil to hold the liquid carrier or a        suspension of the micro spheres in the liquid carrier;    -   c. loading the holding coil with the suspension of the micro        spheres, and with additional liquid carrier for washing the        micro spheres and for rinsing the holding coil;    -   d. providing a porous tube having pores with a pore size of an        appropriate dimension, for retaining the micro spheres within        the porous tube while enabling the liquid carrier in which the        micro spheres are suspended to pass through the pores of the        tube;    -   e. disposing the porous tube in a shell configuration to form a        sequestering cell equipped with an inlet, an outlet, a waste        port, and a flush port;    -   f. with the waste port open and the flush port and outlet        closed, transporting the suspension of the micro spheres to the        porous tube through the inlet to the sequestering cell, for        retaining the micro spheres in the porous tube while passing the        liquid carrier through the pores of the tube;    -   g. washing the retained micro spheres by flowing the liquid        carrier over the retained micro spheres through the inlet to the        sequestering cell to the waste port of the sequestering cell;    -   h. with the waste port and inlet closed and the flush port and        outlet open, dislodging the micro spheres from inner surface of        the porous tube;    -   i. with the waste port and flush port closed and the inlet and        outlet open, withdrawing a suspension of the washed micro        spheres in the liquid carrier from the porous tube;    -   j. transporting the suspension of the washed micro spheres to a        flow cytometer;    -   k. rinsing the holding coil with the liquid carrier; and    -   l. preparing to repeat steps a through k by dispensing liquid        carrier and a bracketing air bubble to the outlet of the        sequestering cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a first and preferred embodimentof a device for sequestering micro spheres, made in accordance with theprinciples of the present invention.

FIGS. 2A, 2B, and 2C are schematic representations of a secondembodiment of a device for sequestering micro spheres, made inaccordance with the principles of the present invention.

FIG. 3 is a schematic representation of a fluidics manifold, made inaccordance with the principles of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

More specifically, reference is made to FIG. 1, in which is shown apreferred embodiment of a coaxial tubular sequestering device, made inaccordance with the principles of the present invention, and generallydesignated by the numeral 2.

The coaxial tubular sequestering device 2 comprises a sequestering cell3 which includes an outer shell 4 and an inner porous tube 6 coaxiallydisposed in the outer shell 4. A plurality of micro spheres 8 suspendedin a liquid carrier 7 are disposed within the inner porous tube 6. Thecoaxial disposition of the porous tube 6 within the outer shell 4minimizes the internal volume of the porous tube 6 while allowingadequate surface area to avoid excessive pressure drop across themembranous wall 6 a as the liquid carrier 7 flows through the membranouswall 6 a of the porous tube 6.

The internal volume of the porous tube 6 is small, typically from aboutfive to about nine micro liters, and is preferably about seven microliters. The pore size of the porous tube 6 is sized to trap the microspheres 8 while allowing the passage of the liquid carrier 7. The microspheres 8 should be larger, preferably about ten times larger, than thepores, and they should not be so small or so numerous as to causeexcessive back pressure in the sequestering device 2. The mean internaldiameter of the porous tube 6 is sized to be compatible with the rest ofa fluid-handling manifold, is typically from about five hundred to aboutseven hundred micrometers, and is preferably about six hundredmicrometers. The thickness of the wall 6 a of the porous tube 6 is fromabout one hundred to about three hundred micrometers, and is preferablyabout two hundred micrometers. The diameter of the micro spheres istypically from about five to about six micrometers. With these typicaldimensions, smaller reagent and wash volumes become feasible.

While the tubular arrangement shown in FIG. 1 is believed to be the mostefficient, other geometries are also feasible. One such arrangement isshown in FIGS. 2A-2C, in which is shown a second embodiment of asequestering device, made in accordance with the principles of thepresent invention, and generally designated by the numeral 14. Thesequestering device 14 comprises a planar porous disk or membrane 10sandwiched between two serpentine passageways 12. The pore size of theporous disk 10 is sized to trap micro spheres while allowing the passageof liquid carrier. The micro spheres 8 should be larger, preferablyabout ten times larger, than the pores, and they should not be so smallor so numerous as to cause excessive back pressure in the sequesteringdevice 2. In a typical application the pore size is from about one-tenthto about three-tenths micrometers, and is preferably about two-tenths ofa micrometer.

Typically, the porous tube 6 and the porous disk 10 are made of porouspolypropylene. Other suitable materials include porous polyethylene,porous polytetrafluoroethylene, Anapore® filter disks, Nuclepore® filterdisks, as well as a wide range of tubular and planar filtration media.

Reference is now made to FIG. 3, in which is shown a fluidics manifold,made in accordance with the principles of the present invention, andgenerally designated by the numeral 16. The fluidics manifold 16 isprogrammed to electronically and automatically control the operation ofthe coaxial tubular sequestering device 2. The fluidics manifold 16comprises a bidirectional pump 16 f, a holding coil 16 e, a firstmulti-port selection valve 16 h, a second multi-port selection valve 16i, an inlet 16 a to the sequestering cell 3, a waste port 16 b for thesequestering cell 3, a flush port 16 d for the sequestering cell 3, anoutlet 16 c to the sequestering cell 3, and a flow cytometer 16 g. Thepump 16 f aspirates a suspension of the micro spheres 8 in the liquidcarrier 7. The holding coil 16 e is used to hold the liquid carrier 7 ora suspension of the micro spheres 8 in the liquid carrier 7. Theselection valve 16 h is fluidly connected to the pump 16 f via theholding coil 16 e, and to the inlet 16 a and the flush port 16 d of thesequestering cell 3. The selection valve 16 i is fluidly connected tothe outlet 16 c and the waste port 16 b of the sequestering cell 3.

To perform a test, the color-coded micro spheres 8, reporter molecules,and sample are drawn into the holding coil 16 e, where they arecombined. This mixture is then dispensed to the porous tube 6, with theoutlet port 16 c and the flush port 16 d closed. The liquid carrier 7passes through the porous tube 6 and to waste via the waste port 16 b.The micro spheres 8 are retained on the surface of the porous tube 6 a.Further reagent and wash solutions may subsequently be pumped over themicro spheres 8 and to waste. To release the micro spheres 8, a smallvolume of the liquid carrier 7 is pumped via the flush port 16 d whilethe outlet port 16 c is open, and the waste port 16 b and inlet 16 a areclosed. In the next step, the micro spheres 8 are drawn back to theholding coil 16 e via the inlet 16 a, with the outlet 16 c open and theflush port 16 d and waste port 16 b closed. The micro spheres 8 are theninjected into an instrument 16 g that uses micro fluidics to align themicro spheres in single file where lasers illuminate the colors insideand on the surface of each micro sphere. Next, advanced optics capturethe color signals. Finally, digital signal processing translates thesignals into real-time, quantitative data for each reaction. Furtherautomation of the sample handling can be achieved by using an automatedfluid handling device to contact the color-coded micro spheres 8,reporter molecules, and sample. To do this, it is convenient to be ableto sequester the micro spheres 8 in a cell at a suitable location in thefluid manifold 16, and then wash various reagents, samples, and washsolutions over the micro spheres 8. The present invention describes anapparatus that fulfills the requirements of such a sequestering cell.

A fundamental design principle for the handling of micro spheres 8 inthe fluidics manifold 16 is to minimize micro-sphere manipulations bybringing the reagents, samples, and wash solution to the micro spheres 8rather than devising complicated micro-sphere transfer sequences. Ourexperience is that micro-sphere manipulation should be kept to aminimum, as micro spheres are easily lost when moving them around innarrow-bore tubing.

The sequestering device should allow for the sequestering of microspheres but allow the passage of liquid. Devices to accomplish this arenot commercially available. The development of such a device is anon-trivial task given the small size of the micro spheres; viz., aboutfive-and-one-half micrometers.

The fluidics manifold 16 is operated according to the following sequenceof steps set forth below in Table 1.

TABLE 1 Fluidic Sequence for Coaxial Tubular Design Step Action 1Aspirate 75 micro liters (μL) of micro-sphere 8 suspension. 2 Load 400μL of liquid carrier 7. 3 Transport micro-sphere suspension to poroustube 6 through inlet 16a at 10 μL/second, with waste port 16b open,flush port 16d and outlet 16c closed. 4 Wash volume of liquid carrier 7over micro spheres 8 through waste port 16b at 10 μL/second. 5 Dislodgethe micro spheres 8 from the surface of the porous tube 6 by dispensing10 μL of liquid carrier 7 from the flush port 16d through the porousmembrane 6a to the outlet 16c. 6 Withdraw micro-sphere 8 suspension fromporous tube 6 at 10 μL/second, with the waste port 16b closed and theoutlet 16c open. Outlet line has previously been charged with flushsolution and a bracketing bubble (see step 8). 7 Transport recoveredmicro spheres 8 to the flow cytometer 16g. 8 Rinse holding coil 16e withliquid carrier 7. 9 Prepare inner porous tube 6 for next run bydispensing 100 μL of liquid carrier 7, preceded by a 25 μL air bubble tothe outlet 16c.

Break-through tests indicate that two-tenths micrometer porous tubing isable to sequester the micro spheres 8. The size of the pores shouldpreferably be much smaller than the diameter of the micro spheres 8, toprevent the micro spheres 8 from becoming lodged in the pores. Next,micro-sphere recovery tests were carried out. The results of these testsare recorded in Table 2.

TABLE 2 Micro-Sphere Recovery from Coaxial Tubular Micro-SphereSequestering Device Volume to Device, μL % Recovery % Std Dev 125 89 5150 101 3 200 91 7 250 92 5 300 97 7

Recoveries were good, and reproducibility was satisfactory.

While certain specific embodiments and details have been described toillustrate the present invention, it will be apparent to those skilledin the art that many modifications are possible within the scope of thebasic concept of the invention. Other applications are not precluded.For example, another field of application is in the separation of plasmafrom red blood cells. The red blood cells will not pass through theporous membrane due to their larger size, but the plasma will.

1. A sequestering device for micro spheres, the sequestering devicecomprising: a planar porous disk having pores with a size with anappropriate dimension to prevent passage of micro spheres while enablinga liquid carrier in which the micro spheres are suspended to passthrough the pores of the disk; and a fluidics manifold, constructed andarranged for electronically and automatically controlling operation ofthe sequestering device, the fluidics manifold including: a firstmultiport selection valve; a bi-directional pump, for aspirating aliquid carrier or a suspension of the micro spheres in the liquidcarrier; a holding coil connecting the first multiport selection valveto the bi-directional pump, for holding the liquid carrier or asuspension of the micro spheres in the liquid carrier; a secondmultiport selection valve; an inlet to the porous disk, wherein theinlet is connected to the first multiport selection valve; a waste portfor the porous disk, the waste port extending from the porous disk tothe second multiport selection valve; and a flush port from the firstmultiport selection valve to the porous disk, wherein the inlet to theporous disk and the waste port from the porous disk define first andsecond passageways, respectively, the first and second passageways areserpentine passageways for causing turbulent flow through thepassageways to suspend and keep suspended the micro spheres in theliquid carrier, and the porous disk is disposed between and fluidlyconnected to the first and second passageways.
 2. A method forsequestering micro spheres, the method comprising the steps of: (a)aspirating, with a flow cytometer, a suspension of the micro spheres ina liquid carrier; (b) using a holding coil to hold the liquid carrier ora suspension of the micro spheres in the liquid carrier; (c) loading theholding coil with the suspension of the micro spheres, and withadditional liquid carrier for washing the micro spheres and for rinsingthe holding coil; (d) providing a planar porous disk having pores havinga pore size of from about one-tenth to about three-tenths of amicrometer, for retaining the micro spheres within the porous disk whileenabling the liquid carrier in which the micro spheres are suspended topass through the pores of the disk; (e) providing the porous disk withan inlet, a waste port, and a flush port, wherein the inlet to theporous disk and the waste port from the porous disk define first andsecond passageways, respectively, the first and second passageways areserpentine passageways, for causing turbulent flow through thepassageways to suspend and keep suspended the micro spheres in theliquid carrier, and the porous disk is disposed between and fluidlyconnected to the first and second passageways; (f) with the waste portopen and the flush port closed, transporting the suspension of the microspheres to the porous disk through the inlet to the porous disk, forretaining the micro spheres in the disk while passing the liquid carrierthrough the pores of the disk; (g) washing the retained micro spheres byflowing the liquid carrier over the retained micro spheres through theinlet to the porous disk to the waste port of the porous disk; (h)charging the flush port of the porous disk with the carrier liquid and abracketing bubble of air; (i) with the waste port closed and the flushport open, withdrawing a suspension of the washed micro spheres in theliquid carrier from the porous disk; (j) transporting the suspension ofthe washed micro spheres to the flow cytometer; (k) rinsing the holdingcoil with the liquid carrier; and (l) preparing to repeat steps (a)through (k) by dispensing liquid carrier and a bracketing air bubble tothe flush port of the porous disk; the above steps being electronicallyand automatically controlled by a fluidics manifold which includes firstand second multiport selection valves, wherein the inlet and flush portof the porous disk are connected to the first multiport selection valve,and the waste port of the porous disk is connected to the secondmultiport selection valve.